Abstract The Eya proteins (Eya 1-4) were initially discovered as essential co-activators of the Six family of homeoproteins that regulate embryonic development via controlling proliferation, survival, epithelial versus mesenchymal fates, and overall cell fate specification in numerous tissues. The Six/Eya complex is downregulated after development is complete, but is re-expressed in numerous cancers, including breast cancer. In addition to acting as transcriptional cofactors for the Six family of proteins, Eyas also contain a Tyr phosphatase activity in their C-terminal domains that has been implicated not only in transcriptional activation, but also in motility, DNA repair and survival in response to damage, angiogenesis, and inhibition of the anti- tumor activity of estrogen receptor-b. Currently, inhibitors are being developed against the transcriptional and Tyr phosphatase activities of Eyas to dissect its different functions, and to potentially develop anti-cancer agents. The Eyas also contain a Ser/Thr phosphatase activity in their N-terminal domain, that is completely separable from the Tyr phosphatase activity. The function of this activity in cancer is completely unknown and it is not targeted by any of the Eya inhibitors under development. Intriguingly, the N-terminal domain of Eya does not contain any recognizable phosphatase motifs and our data suggest that the Ser/Thr phosphatase activity of Eya is not intrinsic, but is instead due to an interaction with the protein phosphatase 2A (PP2A). In addition, using two different immune competent mouse models, we show for the first time that Eya3, which is overexpressed in triple negative breast cancers (TNBC) as compared to other subtypes, and contains the highest Ser/Thr phosphatase activity, influences tumor progression via its effects on adaptive immunity. We further demonstrate that Eya3, through its PP2A-associated Ser/Thr phosphatase activity, regulates the key immune checkpoint protein programmed death-ligand 1 (PD-L1). Thus, we identified a novel tumor promoting function for Eya3 that is only revealed in the context of a functional adaptive immune system. In this proposal we test the hypothesis that Eya3 regulates breast tumor growth and progression via its ability to recruit PP2A, leading to increased levels of PD-L1 and a diminished tumor specific T-cell response. Results obtained in this project will significantly advance our understanding of the role of Eya3 in breast tumor progression, and may also reveal that Eya3, through its previously unknown ability to interact with PP2A, is an important new player in adaptive tumor immunity. Further, we may uncover a novel regulatory mechanism of a key immune checkpoint protein, PD-L1, whose regulation has remained largely elusive. Thus our studies may result in novel biomarker or therapeutic strategies to enhance efficacy of PD-1/PD-L1 mediated therapies.